Composition containing and method of using a fibrinolytic active principle

ABSTRACT

A natural active principle enzymatic in type, called angiokinase, is obtained with an extraction procedure starting from mammalian connective tissue, such as from cattle and the like. The active principle comprises a new drug for the prophylaxis and treatment of all symptoms of abnormal fibrin formation and thrombus formation in various sites and localizations, such as atherosclerotic diseases and diabetic retinopathy, as well as thrombosis and thromboembolisms.

This is a divisional of co-pending application Ser. No. 366,276, filedApr. 7, 1982 now U.S. Pat. No. 4,471,053.

The present invention refers to an extraction process for the productionof a vasal fibrinolytic active principle starting from mammalianconnective tissue, as well as to the specific enzymatic product soobtained, called `angiokinase.` The invention moreover relates to theuse of said product in pharmaceutical formulations for the preventionand treatment of diseases caused by abnormal fibrin and thrombusformation in various sites and localizations.

BACKGROUND OF THE INVENTION

The principle object of this invention is to provide a new naturalextractive principle, enzymatic in type, with fibrinolytic action invivo, both direct and through plasminogen activation. Since saidprinciple is obtained from the highly vascularized connective tissue ofmeat animals, it may be named simply and unequivocably `angiokinase` inanalogy with urokinase, the proteolytic plasminogen-plasmin activatorisolated from the urine of male human subjects (see U.S. Pat. Nos.2,961,382; 2,983,647; 2,989,440; 3,081,236; see also White et al.,Biochemistry, 5, 2160, 1966). Another analogous name is `streptokinase,`another plasminogen activator isolated from hemolytic streptococcuscultures (see U.S. Pat. Nos. 3,016,337; 3,042,586; 3,063,913; 3,063,914;3,107,203; 3,138,542; 3,276,304).

The significance and the precise meaning of this invention, as well asthe utility of its application to the prevention and cure of venal andarterial thrombosis in human subjects will be more readily appreciatedafter a brief discussion of the currently accepted and widely known viewthat mammalian blood vessel walls show fibrinolytic action. Said actioninvolves the production of principles specific to this activity,currently known as asminogen activators. Early views were that thefibrinolytic activity of the vasal wall would predominantly involve theveins and small vessels, but later the activity was observed in largevessels as well, in particular in the wall of the aorta. Research hasshown that the blood vessel walls in human subjects killed in accidents(after incanulation of the femoral artery and vein) contained aconsiderable quantity of so-called `plasminogen activators.`

There was no effective conclusion to the above studies regarding theisolation by chemico-physical extraction of a particular activeprinciple from the vasal wall and from other highly vascularizedconnective tissues. Therefore, the present invention proposes theisolation and characterization of the active fibrinolytic principle fromblood vessels in general and from the aorta in particular, calledangiokinase, previously unknown. The invention also relates to itsclinical use for therapeutic indications as a general antithrombotic andalso within certain limits as a platelet anti-aggregant. In fact, thepresence of angiokinase due to the onset of fibrinolytic activity on thepart of the walls and then the blood, provides a first level ofresistance to thrombosis conditions in warm blooded animals, includinghumans.

Thus the substance angiokinase, isolated and described in thisinvention, is the fibrinolytic principle in vasal walls in general andin the aorta in particular, which may be re-added to circulating bloodto provide said blood with fibrinolytic potential.

Angiokinase shows the important property that it acts both as anactivator (plasminogen plasmin) and as an actual plasmin. This findingis well documented, as described below in support of the invention, andis of considerable theoretical as well as practical significance. Upuntil now, all studies of the fibrinolytic activity of vasal walls haveonly revealed the simple activation effects of the pre-formedplasminogen in circulation. Angiokinase however also acts directly inexactly the same way as plasmin.

The areas of use of the present invention will be discussed in detailbelow.

As is well known, an event of particular importance in circulatingpathology, at both venal and arterial levels, is the endovasal depositof fibrin--said event leads both to stenosis of the vasal lumen and toocclusion (thrombosis).

Fibrin deposition also plays a particularly important role in whiteplatelet thrombus formation, since it is required for adhesion of theplatelets to the vasal wall and for platelet aggregation as well as forstabilization of the platelet aggregate.

Keeping the interface between blood and vasal wall free is an importantand fundamental process in physiopathology.

It is also well known that the endovasal occlusive event (stenosis andthrombosis) is an indispensable and determining factor for a clinicallynon-detectable blood vessel condition to become visible on thesymptomatic level and to thus become a clinically manifest disease.

This is quite freqnently the case in the athero-arteriosclerotic processwhich in itself is clinically invisible but becomes evident on theclinical level only (or mainly) when the endovasal stenosis process isaccentuated or superceded by actual occlusion.

Therefore, for both prevention and therapy, it is of prime importance toon the one hand block and prevent the formation and accentuation offibrin deposits and on the other to dissolve these deposits after theyare formed.

As is well known, to this end therapy must be instituted to dissolve thefibrin which is forming and the fibrin deposits already formed.

This therapy thus involves the known products with fibrinolyticactivity.

The medical profession is in complete agreement regarding thesignificance and importance of fibrinolytic therapy: it assumes aprimary role in circulatory pathology.

There are two conditions underlying fibrinolytic therapy; The first isthat, as mentioned above, fibrin is a determining factor in stenosis andendovasal occlusion.

The second is that a deficit in fibrinolytic activity in theblood-vascular system is a pathogenetic factor of prime importance notonly in conditioning for the stenosis and vasal occlusion process butalso in the same conditioning for atherosclerotic disease.

Too little capacity in the production or release of fibrinolyticactivity by the vasal wall is an important factor in determining theatherosclerotic process in general and the endovasal occlusive processin particular. Therefore, a deficit in blood-vasal fibrinolytic capacityhas considerable importance for the apthogenesis of atheroscleroticdisease as well as for determining its most severecomplication--thrombosis.

It is also well known that tissues rich in blood vessels, arterial andvenous, are able to produce and release plasminogen activators and thusto show fibrinolytic activity. This then is a tissue activator ofplasminogen, meaning the production of fibrinolytic activator at bothtissue and vasal levels.

Using the methods of tissue extraction, histo-chemistry and in vitrotissue cultures, tissue and vascular fibrinolytic activity has beendemonstrated and widely confirmed, and even measured at natural levels.It is known that tissue activators can be isolated from the heart, lung,ovary, prostate, uterus, etc.: these tissue activators are currentlyknown as TA.

The plasminogen activator, which plays the most important role inintravascular fibrinolysis, is thought to be that synthesized by theendothelial cells topping the blood vessels. Since this enzyme has notyet been isolated and characterized, it has not yet been possible toidentify this vascular activity with that of the tissue activator, eventhough these activities may really be the same.

The vascular or endothelial activator has been the object of numerousstudies, and has been recognized and demonstrated, as well as evenisolated, from the experimental medicine and biological activity pointsof view. The plasminogen activator has even been isolated anddemonstrated from the vascular tissues of human cadavers.

However, it should be noted that during the thrombotic event, botharterial and venous, the capacity for fibrinolytic production is indeficit and compromised.

Another significant fact to be considered is the reason why this is heldto be very important not only for the thrombotic complications arisingduring the course of ongoing vasculopathies (for example,athero-arteriosclerotic and varicose complications) but also for theirpathogenesis, as already mentioned for the presence of a fibrinolyticdeficit of the vasal wall.

The lack of plasminogen activators characterized atheroscleroticdisease.

A significant correlation has been noted between reduced fibrinolyticactivity by the arterial wall and susceptibility to atherosclerosis. Adeficit in this activity has been found to be directly proportional togreater and more severe susceptibility and sensitivity toexperimentally-induced atheromatous processes.

Moreover, administration of antifibrinolytic agents aggravates theatheromatous process, while administration of fibrinolytic agentsattenuates it.

Likewise, administration of antifibrinolytic drugs increases the risk ofthrombotic complication, and the risk of systemic thromboticcomplications must be taken into account when antifibrinolytic drugs areadministered.

On a clinical level, a deficit of fibrinolytic activity has also beenshown in atherosclerotic disease, which could be related mainly to adeficit in the production and release of plasminogen activators by thevasal wall and its endothelium in particular.

In atherosclerotic vascular conditions, a real incapacity and reducedpower of fibrinolytic response by the vasal wall has been found.

Another particularly important finding has been a deficit of localfibrinolytic activity, or rather of a deficit predominantlycorresponding to the vascular regions affected by the atheroscleroticprocess as compared to less affected and undamaged areas.

With regard to the human myocardium after suffering an infarction(extracts from myocardial tissue soon after an infarction), a sharpreduction or absence of fibrinolytic activity has been found. However,myocardial tissue extracts from infarcted areas in the fibrosis andconnective repair phase show said activity.

Analogous results were found in experimentally-induced myocardialinfarction: the fibrinolytic activity previously lacking was then foundin lesioned areas undergoing repair, because of the plasminogenactivators in the newly formed blood vessels.

Another significant finding is that of a fibrinolytic compromission inocclusive venal conditions. Fibrinolytic activity is reduced andcompromised in the wall of the thrombus-affected vein. Low fibrinolyticactivity in the vasal wall was found to be associated with an increasedtendency to develop thrombosis in the deep veins. Thus a largepercentage of a large series of patients with severe thromboembolicdisease showed decreased fibrinolytic activity of the vasal wall.

The correlation between low fibrinolytic activity of the vasal wall andthe blood on the one hand and the onset of thrombosis or pre-thromboticconditions on the other has been confirmed in several studies.

Diabetes melitis, especially in combination with obesity, has beenassociated with reduced vascular and/or blood fibrinolysis due tostasis.

It is also known that obesity increases the predisposition of diabeticsto vascular occlusions.

With regard to another situation favouring thrombosis development,depressed fibrinolytic activity in the venal wall, and/or reducedrelease of fibrinolysis activators be the vascular wall, has been foundto be a regular post-operatory phenomenon, one that is particularlydepressed in those patients who developed thrombosis.

Also, immediately after serious operations, the vascular endotheliumcannot respond to stasis as it could before the operation. The term`endothelial decompensation` has been suggested to explain the decreasein fibrinolytic activation in the post-operatory period.

Reduced tissue fibrinolysis is thought to be implicated inpost-thrombotic syndrome, with consequent insufficient blood supply tothe skin and ulceration.

These clinical and experimental data show that parietal plasminogenactivator is greatly decreased in thrombinogenesis, and this observationsuggests that these agents are consumed in the attempt to resolve thethrombosis.

Further evidence for the antithrombotic action of the vascularplasminogen activator was obtained when it was found that the greatestdanger of experimentally-induced occlusion (for example, during thefirst eleven days after an operation) generally coincided with reducedor even absent fibrinolytic activity by the vascular wall.

All of the known data in this field have convinced experts in the areathat adequate fibrinolytic treatment is an important part of humantherapy in the indicated cases.

DESCRIPTION OF THE PRIOR ART

The area of the present invention, in relation to the improvement itaffords over previous techniques, may be described in the followinggeneral terms.

Currently available fibrinolytic activators are well known to be of theindirect or direct types.

(a) Indirect means

These are not part of the actual fibrinolytic system, but ratherstimulate the tissues (for example, vasal wall) to produce fibrinolyticactivators (for example, stanazolol, ethyl estrenol, penphormin) or canmobilize the already produced activators from the wall (for example,nicotinic acid and its derivatives, acetylcholine, etc.).

The former show more continuous fibrinolytic activity which is howeverrather modest and somewhat slow--sometimes months of treatment areneeded before any effects are seen.

The latter show more rapid and pronounced action, which however iscompletely transitory and followed by periods of reduced fibrinolyticactivity during which the organism is more susceptible to thrombosis.

(b) Direct means

These form part of the fibrinolytic system itself, and involve both STK(streptokinase) and UK (urokinase) mentioned above. These are in effectthe agents almost always used.

It must be noted that while these agents can develop prompt andcontinuous fibrinolytic activation, they are still far from satisfyingphysiopathological and clinical requirements. In particular, STK suffersthe handicap of antibody inactivation. Moreover, both STK and UK providefibirnolytic activation which leads to plasmin formation, which is inturn inactivated by antiplasmins. In fact, the fibrinolytic activationinduced by STK and UK, and thus the consequent plasmin formation (theessential condition for their effective action), runs counter to theinhibition caused by the antiplasmins in circulation and in particularby the fast-acting alpha₂ antiplasmin. This fact leads to theneutralization of a given part of these activators, and so necessarilyto the use of elevated dosages to overcome this inhibition, withconsequent risk of hemorrhage.

Moreover, UK differs from the tissue activator and thus from thevascular plasminogen activator in that it forms part of fibrin-freeurinary system and is thus extraneous to the blood vessel regions.Therefore, UK shows low affinity for fibrin, unlike the vascularplasminogen activator with its fairly pronounced activity in thisregard. Synthesized by the kidney and isolated from the urine, UK islocalized in the urinary tract where it has no effect on intravascularfibrinolysis.

OBJECT OF THE INVENTION

The object of the present invention is a process the isolation andpreparation in therapeutically suitable form, and a tissue plasminogenactivator obtained via an extraction process from richly vascularizedmammal organs and tissues.

Given its enzyme-type activity, said activator has been named`angiokinase` in analogy with urokinase and streptokinase.

The tissue plasminogen activator, including that from the vasal wall(also known as `endothelial activator` since it is produced by thevascular endothelia), differs from urokinase and streptokinase in itsaffinity for fibrin. This phenomenon was used in its isolation, asdescribed below.

Endothelial plasminogen activator is readily adsorbed by the fibrinsurface, facilitating plasminogen activation in this site. Theplasminogen activator present in the blood is very probably identical tothe vascular plasminogen activator that is object of this invention. Inturn, the plasminogen activators in the tissues are probably verysimilar or identical to the blood and vascular activator.

Prior to this invention, the tissue activator (endothelial) has not beenavailable for therapy even though it was hypothesized to be the mosteffective.

Vascular plasminogen activator is tightly adsorbed by the fibrin. Thefibrin site which fixes the plasminogen is the same as that which fixesthe vascular activator. In this way, a single mixture is formed ofplasminogen, its activator and the fibrin to form plasmin on the insideof the thrombus.

In this way, the plasmin site which fixes it is rapidly occupied by thefibrin, to then protect the plasmin from inhibition by alpha₂antiplasmin. STK and UK, however, as mentioned above, are subject toantiplasmin inhibition.

It is thus obviously quite important to have available for therapy thecorrect form of the tissue plasminogen activator according to thisinvention, in particular the vascular or endothelial activator.

The active fibrinolytic principle according to this invention waspreviously known only in the limited terms of its supposed existencebased on its biological effects. It was called angiokinase since itcomes from the vasal wall, or in any case from rich vascularizedtissues.

Its undoubted biological activity has been confirmed based on amechanism of action particularly suited to its physiopathological andtherapeutic ends.

The active principle of this invention offers the following advantagesover current practice, i.e., the use of STK and UK:

1. Precise, rapid and significant fibrinolytic activity.

2. This fibrinolytic activity has been shown to be of the binary type,since the active principle shows both activator type and plasmin typeactivity.

3. The resulting plasmin activation is insensitive to plasmaantifibrinolytic agents, and in particular to alpha₂ antiplasmin.

4. No antigen effect.

5. High fibrin affinity.

6. No risk of hemorrhage within a wide dosage range, and thus excellenttolerance.

7. Exact measurement in units of fibrinolytic activity.

Table I below compares the therapeutic use of STK and UK on the one handwith that of angiokinase (AK) on the other.

                                      TABLE I                                     __________________________________________________________________________    Streptokinase and Urokinase                                                                            Angiokinase                                          (STK)(UK)                (AK)                                                 __________________________________________________________________________      Antibody inactivation (STK)                                                                        1.                                                                              No antibody inactivation                               Plasmatic antiplasmin inactivation                                                                 2.                                                                              No inactivation by plasmatic inhibitors                Limited or scarse affinity for fibrin                                                              3.                                                                              High affinity for fibrin                               Extraneous to blood coagulation homeo-                                                             4.                                                                              Legitimate part of blood coagulation                   stasis and to the relative fibrinolytic                                                              homeostasis and the relative fibrin-                   mechanism              olytic mechanism                                       Risk of hemmorrhage  5.                                                                              No risk of hemorrhage                                  Poor correspondence between dose and                                                               6.                                                                              Good correspondence between dose and                   effect                 effect                                                 Activating action only                                                                             7.                                                                              Binary action: activating and plasmin                  Intravenous administration (phlebo-                                                                8.                                                                              Intravenous, intramuscular and oral ad-                clysis)                ministration                                           Limited administration period                                                                      9.                                                                              Prolonged and repeated administration                                         for any period of time                               __________________________________________________________________________

It should be noted that the binary fibrinolytic action of angiokinaseoffers a considerable advantage in both absolute and relative (to STKand UK) terms: it allows fibrinolytic activity even in the deficit orabsence of plasminogen, that is, in situations where an activator onlyis ineffective.

In turn, the lack of inhibition by plasmatic inhibitors guarantees asatisfactory correspondence and proportionality between dosage and theresulting effect.

Clearly, the discovery of the tissue (vascular) fibrinolytic activeprinciple, that is of angiokinase, which is the object of the presentinvention represents important and real progress in the field offibrinolytic and thrombolytic therapy. Said progress allows the use ofthe same fibrinolytic principle directly involved in coagulationhomeostasis in the blood vessels.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is a process for theproduction of a vasal fibrinolytic active principle called angiokinase,starting from richly vascularized connective tissue from mammals,particularly the aorta of animals like cattle and pigs, where saidprocess involves the following operations:

defatting, drying and pulverizing said tissue;

subjecting said powder to pancreatin lysis by forming an aqueoussuspension of it at substantially neutral pH, adding approximately 1% byweight of pancreatin with respect to the weight of powder used, and byeffecting pancreatin digestion at a temperature of 45°-50° C. forapproximately 24 h with stirring, so as to prepare a lysate suspension;

acidifying the suspension to bring the lysate into solution and form aprecipitate which is then removed from the solution by filtration;

neutralizing said solution and adding a sufficient quantity of ammoniumsulfate to reach an ammonium sulfate concentration of approximately 35%of saturation, so as to precipitate said lysate;

collecting the precipitate containing the active principle and formingan aqueous suspension at neutral pH;

purifying said lysate suspension through selective fractionationoperations which may be of several types, for example, through selectiveelution of a gel column followed by purification via selectivefractionation via chromatography, or through elution using a largevolume of gel in proportion to the liquid to be purified, leading to adilute solution of the active principle which is then concentrated byremoving some of the water; and collecting the fractions containing theangiokinase, checking the activity of said fractions in fibrinolysis.

Also an object of the present invention is the vasal fibrinolytic activeprinciple called angiokinase, obtained via the procedure mentionedabove, with a protein N content of 0.2 μg/ml and a mean molecular weightof about 18000 Dalton.

Another aspect of this invention is the application of angiokinase inhuman therapy for the prophylaxis and treatment of abnormal fibrinformation and of thrombus formation in its various manifestations.

The procedure for the preparation of the active principle is thuscharacterized by the choice of starting materials consisting of richlyvascularized connective tissues to obtain a product containing theactive principle in highly concentrated form, along with mainlyproteinic impurities. Thus, successive purification is necessary toremove the undesired impurities.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description below refers to the attached drawings, inwhich:

FIG. 1 is a diagram showing the fibrinolytic activity of the fractionsobtained from the lysate during the first purification phase, usingadsorption chromatography;

FIG. 2 is a diagram showing the fibrinolytic activity of the fractionsobtained from the lysate during the second purification phase, usingadsorption chromatography;

FIG. 3 is a spectrum used to check the adsorption chromatographicpurification of the lysate;

FIG. 4 shows the fibrinolytic activity of the fractions obtained fromthe lysate in the second purification phase using ion exchangechromatography;

FIGS. 5 and 6 are diagrams representing angiokinase's plasminogenactivation in experiments with short and long incubation periods,respectively.

THE DETAILED DESCRIPTION OF THE INVENTION Preparation procedure

The starting material for the angiokinase extraction consists of variousmammalian organs, particularly organs and connective tissue from cattleand pigs which have a high proportion of vascularized tissues.

According to the present invention, angiokinase was isolated andidentified both from arterial and venal vasal tissue and from highlyvascularized organs and tissues, in which it was present in quantitiessufficient for extraction.

Examples of organs and tissues used for angiokinase extraction includethe aorta, heart, lung, spleen, liver, pancreas, although the aorta ispreferred, although the invention is not limited to these organs only.

The starting material is first treated to remove fats, for example byextraction with a solvent like acetone, and then dried, reduced topowder and subjected to pancreatin lysis. Lysis is performed byresuspending the dried organ in aqueous solution at neutral pH, forexample in pH 7.4 phosphate buffer, and adding industrial pancreatin ina quantity equal to approximately 1% by weight with respect to the organin suspension. Lysis is carried out by digestion at a temperature of45°-50° C., preferably with constant stirring, for approximately 24hours.

After the digestion, the insoluble portions are removed bycentrifugation and the liquid is collected and acidified to pH 2.5,preferably with dilute HCl, until a precipitate is formed. Theprecipitate is again separated by centrifugation, and the liquidcollected is neutralized to pH 7 by adding dilute NaOH.

The active component is separated from this neutralized liquid by addingammonium sulfate ((NH₄)₂ SO₄ ) until the concentration reaches 35% oftotal saturation. In this way a precipitate is formed which contains theactive fibrinolytic component of the organ lysate.

The precipitate is collected by centrifugation and the liquid isdiscarded. The precipitate is then resuspended in a small volume ofphosphate buffer at pH 7.4.

The resulting suspension is already a concentrated extract of the activeprinciple angiokinase.

Various reproducible industrial methods may be used to effect thispurification; the following two are cited as examples only.

The first method involves a two step purification: the first by gelfiltration to give a partial purification and the second byfractionation via adsorption or ion exchange chromatography.

The second method consists of an operation analogous to the first stepabove, but with using higher ratios of gel to product to be purified soas to achieve greater purification in a single step. The resultingproduct is very dilute and requires successive concentration by removingthe excess water.

In order to describe the invention in greater detail the followingexamples describe the operations carried out in purifying the lysateprepared as described above.

EXAMPLE I First partial purification step of the lysate, using SephadexG-25 filtration gel

This is a simple and rapid method for the partial purification of largequantities of lysate prepared as described above, leading to goodfractionation of the proteins based on their molecular weight. Alongwith other large molecules, the active principle is completely excludedby the gel and so is eluted immediately, while spherical globules belowthe highest fractionation limit (from 1000 to 5000 dalton, for theSephadex G-25 used in this example) are eluted later.

This method also desalts the lysates, through combination with thebuffer.

The advantage of this method is that it allows the filtration of largevolumes: volumes of the substance to be eluted can be up to 30% of thevolume of the gel. For example, approximately 300 ml of the organ lysatemay be used for each liter of gel.

The following experimental conditions were used: 10 ml of bovine aortalysate is placed on a column (2.6×40 cm) with Sephadex G-25 (60 ml gel,33 ml/hour flow rate, approximately 5 ml every 9 minutes) in 0.1 M TRISbuffer (trihydroxymethylaminomethane), treated with 0.02 M CaCl₂ at pH8.2.

The eluent is collected in 5 ml fractions. The fractions containing theactive principle are checked by measuring their fibrinolysis activity,using the method described below. Under these experimental conditions,the active principle is eluted in the first eight fractions.

FIG. 1 shows a graphical representation of the typical progress in thisfirst partial purification of the organ lysate.

In FIG. 1, the fraction number is reported along the ordinate while thelysis area in mm² is reported along the abscissa (measured as describedbelow).

Using the same methodology with a different buffer gives an analogouspartial purification, for example using phosphate rather than TRISbuffer (again at pH 8.2), or 0.1 M sodium acetate buffer(at another pH45.0). These and other variations are well known to experts in thisfield. Before going on to the second step to achieve a high degree ofpurity of the active principle, a dialysis process must first be carriedout. As mentioned above, after the first partial purification step, theangiokinase can be prepared using various purification methods. Thefollowing examples report only those with particularly advantageousresults, although other methods may of course be used without goingbeyond the bounds of the present invention.

EXAMPLE II Second step--total purification of angiokinase usingadsorption chromatography

The enzyme angiokinase can be purified via adsorption chromatographyusing various chromatographic substances, for example:

(A) Soy bean trypsin inhibitor (SBTI) bound to Sepharose

(B) p-Aminobenzamidine (p-ABM) bound to Sepharose.

II-(A) Angiokinase purification by adsorption chromatography with soybean trypsin inhibitor (SBTI) bound to Sepharose

In adsorption chromatography, the substrate Sepharose (well known to beproduced and sold world-wide by the Pharmacia Fine Chemicals Company,Uppsala, Sweden) bound here to SBTI, has affinity for the object of thepresent invention, angiokinase. This is due to the covalent bondformation between the activated substrate and the enzymatic protein,which is thus adsorbed from solution.

It is then de-sorbed by varying the pH, or ionic strength, of themedium, after the inert unbound material is eluted.

Preparation of SBTI-Sepharose

7.5 g of Sepharose 4B is washed on glass wool with a 0.001 M NaClsolution (pH 8.3).

The washed gel is suspended in the above buffer, and stirred gently forapproximately 2 hours at 4° C. Excess bonding agent is removed, byalternate washes with higher pH (0.1 M TRIS+1.0 M NaCl; pH 8.0) andlower pH (0.1 M NaAcetate+1.0 M NaCl; pH 4.0) buffers. At the end thegel is obtained in the more acid buffer, ready for use.

FIG. 2 gives a graphical representation of the adsorption chromatographyof 10 ml of active bovine aorta lysate fractions (partially purified asdescribed above with Sephadex G-25 in 0.1 M TRIS+0.02 M CaCl₂ buffer, pH8.2). The fraction number is reported along the abscissa, while thelysis area in mm² is reported along the ordinate (measured as describedbelow).

The progress of the adsorption chromatography was followedspectrophotometrically, at 280 nanometers in this case. The initialbuffer was that mentioned above (pH 4.0). When the extinctioncoefficient at 280 nm reached 0, the buffer was replaced with one at pH2.0 (0.2 M KCl 0.2 M HCl). The flow rate was approximately 3-5 ml/5minutes. Every test tube collected was tested by UV.

As shown in FIG. 2, a sharp protein peak is obtained, coinciding withthe onset of fibrinolytic activity, between the 40th and 48th fractions,corresponding to the isolation of angiokinase.

Angiokinase may also be purified starting directly from the organlysate--without the partial purification described in example I.However, in this case the lysate must be dialyzed with the same buffer.

(II-B) Angiokinase purification by absorption chromatography withp-aminobenzamide (p-ABM) bound to Sepharose

The principle is as described above in example II-A.

Preparation of Sepharose with bound p-ABM

6 g of Sepharose 4B is weighed, and the gel is washed on a glass filterwith 1000 ml of 0.5M NaCl (reagent A).

90 mg of N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide HCl isdissolved in 4 ml of distilled water and brought to pH 3.9 with 1 ml of0.01M HCl (reagent B).

90 mg of ρ-aminobenzamidine is dissolved in 4 ml of distilled water atpH 4.5 (reagent C).

The three reagents (A,B,C) are mixed together to give a solution withfinal pH around 4.8 which is then stirred for 20 hours at roomtemperature. The resin is then washed with twice distilled water.

7 ml of Sepharose bonded to p-ABM is equilibrated with 4-5 volumes of0.1M sodium phosphate at pH 7.6 in a 1 cm diameter column. The solutioncontaining the angiokinase enzyme is then placed on the column (100 mlof partially purified organ lysate, brought to pH 7.6 with 0.1M TRISbuffer, are needed to saturate a column of 7 ml of Sepharose-p-ABM).

An aliquot of the lysate is kept aside to measure the activity.

Activity is measured before and after the partially purified lysate(example I) is passed through the bonded resin (measured by fibrinolysisor chromogenesis, as described below) to show when the column issaturated, i.e., when the activity is identical.

The column is then washed with 0.1M sodium phosphate buffer at pH 7.6until the monitor shows 5% transmission, where the monitor is acontinuously reading spectrophotometer set at 280 nanometers.

The column is then eluted with 0.1M benzamidine-HCl dissolved in thesame buffer.

The samples to be collected (4-5 ml) are those just after the opticaldensity (O.D.) of the benzamidine effluent reaches the maximum value ofapproximately 90% absorbance.

The samples collected are dialyzed with 0.1M sodium phosphate buffer atpH 7.6 in a cold chamber at 4° C. with continuous stirring for 24 hours,with frequent changes. In this way, the benzamidine is removed and thedialyzed samples are frozen after the activity is measured (as describedbelow).

FIG. 3 shows the spectrophotometric curve for the chromatographycontrol, referring to the present example. % optical density is reportedalong the abscissa and fraction number along the ordinate.

EXAMPLE III Second step--complete angiokinase purification using ionexchange chromatography

In ion exchange chromatography, the gel contains negatively orpositively charged groups. If the gel is positively charged (anionexchange), the negatively charged components of the solution to bepurified are adsorbed. By using an eluent buffer of higher ionicstrength, the proteins adsorbed are replaced by negative ions from thebuffer (salt gradient). Each type of protein is selectively eluted atits specific gradient. This method allows complete purification of theangiokinase in the organ lysate treated as in example I;

Sephadex CM-50 (Pharmacia Fine Chemicals) has been found to be the idealion exchange resin for this purification.

The active fractions (from the 6th to 14th test tubes, for a total of 30ml) of lysate partially purified by column chromatography on SephadexG-25 (gel filtration as in example I) are combined and passed through a24×188 mm column containing Sephadex CM-50 (60 ml) with 0.1M sodiumacetate buffer at pH 5.0.

If the fractions of lysate partially purified on Sephadex G-25 are in pH8.2 buffer, they must be dialyzed and taken up in pH 5.0 acetate buffer.This is not necessary if the partial purification of example I has beendone directly at pH 5.0.

The flow is regulated to 16 ml/hour, and the fractions are collectedwith 5 ml/test tube. The progress of the chromatography is followedthrough continuous spectrophotometric reading at 280 nanometers as wellas by measuring the fibrinolytic activity as described below.

The gradient is prepared by mixing 150 ml of 0.1M sodium acetate bufferwith 150 ml of 0.1M sodium acetate +0.6M NaCl buffer.

FIG. 4 gives a graphical representation of angiokinase purification viaion exchange chromatography, as described in this example. Fractionnumber is reported along the ordinate, and lysis area in mm² along theabscissa.

The curve shows a first sharp peak corresponding to a low activityproduct. When the absorbance has returned to zero (from the 30th to 60thtest tube), the molarity of the buffer is increased using the samesodium acetate buffer containing 0.6M NaCl (gradient application). Inthis way, ionic strength is increased, while the pH remains fixed at5.0.

Under these conditions, other protein material passes (low peak, flatabsorbance) corresponding to a high peak of activity. These are thefractions containing the purified angiokinase.

EXAMPLE IV Complete angiokinase purification in a single step, usingcolumn chromatography with Sephadex G-25

This method is conceptually the same as that described in example I forthe partial purification of the organ lysate from which angiokinase isextracted.

By varying the experimental conditions, that is by varying the ratio ofgel volume to that of the substance to be chromatographed, the enzyme tobe isolated may be purified even further. In other words, by greatlyincreasing the gel volume with respect to that in example I and byreducing the flow rate, the various fractions eluted may be partitionedmore precisely to give fractions of angiokinase of a high degree ofpurity.

However, this operation must be followed by one in which these activefractions are concentrated, for example, via lyophilization,concentration at reduced pressure, and the like, since the enzyme iscollected from the column in a highly dilute solution.

Properties of the product

Once the purified enzyme is prepared using the methods described above,it is then identified and characterized.

A preferred, but non-limiting, example of this process is a purifiedlysate from bovine arterial wall, prepared via pancreatin lysisaccording to this invention.

The following materials were used to evaluate the product, using themethods described below:

suine plasmin, activated with trypsin (Lysofibrin, Novo Laboratories,Copenhagen); 2 units of NOVO dissolved in 0.15M NaCl immediately beforeuse;

human plasminogen, purified chromatographically from the plasma,supplied by the KABI Company, Stockholm, 15 U/mg specific activity;

human fibrinogen, prepared from Cohn fraction I, supplied by the KABICompany, Stockholm, dissolved in 0.1M TRIS buffer, pH 7.8;

thrombin (Topostasin, Roche, Basil), dissolved in 0.15M NaCl immediatelybefore use;

Sephadex, CM-50, G-75 and G-100, supplied by the Pharmacia FineChemicals Company, Uppsala Sweden.

The results were compared with a previously known fibrinolytic agent:

Urokinase, supplied by the Leo Company, Copenhagen, in injectable form,in ampuls of 7100 CTA units each, dissolved in 0.15M NaCl immediatelybefore use.

The following methods were used for data determination:

(1) Fibrinolytic activity was measured using Astrup and Mullertz' fibrinplate method, modified as described below (see `Titration` section).Plasminogen-rich and plasminogen-free fibrin plates were used. Thefibrinogen on the plasminogen-free plates was coagulated withplasminogen-free thrombin.

(2) The protein content was measured using the method of Lowry et al.

(3) Molecular weight was determined using 2.5×100 cm Sephadex G-100columns, calibrated with Dertrane Blue (Vo) and K₂ CrO₄ (Vt). CytochromeC, ovoalbumin, bovine serum albumin and transferrin were used ascontrols.

(4) Electrophoresis on polyacrylamide gel: the sodium dodecylsulfate geland buffers were prepared according to Weber and Osborn (1969) with andwithout beta-mercaptoethanol.

Acrylamide concentration in the gel was 10%. The gels were stained forproteins with Camassie Blue, or sliced into 1.0 mm thick disks with aslicer.

The disks were tested for fibrinolytic activity by placing them directlyon the fibrin plates.

(5) Method for detecting possible plasminogen activation by the purifiedextract:

1.6 mg of plasminogen is dissolved in 2 ml of distilled water, anddialyzed for 12 hours with 0.01M sodium phosphate buffer (pH 7.0). Asmall quantity of undissolved material is removed by centrifugation at4000 g at a temperature of +4° C., for 20 minutes. The plasminogensolution is diluted with phosphate buffer to a final volume of 2 ml. 1ml of purified extract (angiokinase, 0.025 extinction) is dialyzedanalogously at the same time.

An ampul of urokinase is dissolved in phosphate buffer to the desiredconcentration.

The plasminogen is then incubated with the purified extract or urokinasein the following ratios:

0.3 ml plasminogen solution+0.1 ml angiokinase solution

0.3 ml plasminogen solution+0.1 ml urokinase solution

0.3 ml plasminogen solution+0.1 ml buffer.

The incubation period is two hours at +37° C. The reaction is quenchedby adding a small quantity of sodium dodecylsulfate and 2 μ-liters ofbeta-mercaptoethanol in each test tube. 100 μ-liters of each of thesesolutions is then examined by electrophoresis on polyacrylamide gel.

Alternatively, the test solution may be incubated as follows:

0.3 ml plasminogen solution+0.1 ml glycerin+0.1 ml AK

0.3 ml plasminogen solution+0.1 ml glycerin+0.1 ml UK

0.3 ml plasminogen solution+0.1 ml glycerin+0.1 ml buffer.

The incubation period is 30 minutes, after the solutions are examined asabove.

Results

The following results were obtained from the measurements describedabove:

(1) The purified enzyme (angiokinase) is active on both theplasminogen-rich and plasminogen-free plates.

(2) The protein content of the purified enzyme (ion exchange) is N=0.2μg/ml.

(3) The mean molecular weight of the angiokinase protein isapproximately 18000 dalton.

(4) The electrophoresis results are consistent with chose for directfibrinolytic activity.

(5) Since both the starting extract and the purified principle wereactive on the fibrin plates both with and without plasminogen, theactive principle was tested to determine whether it has plasmin activityonly or a plasminogen activation effect as well. A preliminary test iswhich plasminogen was incubated in increasing concentrations in thepresence of purified extract, showed that the fibrinolytic activityincreases with the plasminogen concentration. This shows thatangiokinase has a plasminogen activating effect. The data from thisexperiment are reported in table II.

                  TABLE II                                                        ______________________________________                                        Angiokinase fibrinolytic activity as a function of                            plasminogen concentration                                                                             Fibrinolytic activ-                                   Solutions               ity (mm.sup.2 lysis)                                  ______________________________________                                        0.5 ml AK + 0.5 ml plasminogen 5.00 U/ml                                                              264.253                                               0.5 ml AK + 0.5 ml plasminogen 2.50 U/ml                                                              170.176                                               0.5 ml AK + 0.5 ml plasminogen 1.25 U/ml                                                              136.130                                               0.5 ml AK + 0.5 ml plasminogen 0.75 U/ml                                                              104.108                                               0.5 ml buffer + 0.5 ml plasminogen 5.00 U/ml                                                          22.22                                                 0.5 ml buffer + 0.5 ml plasminogen 2.50 U/ml                                                          trace                                                 0.5 ml buffer + 0.5 ml plasminogen 1.25 U/ml                                                          0.0                                                   0.5 ml buffer + 0.5 ml plasminogen 0.75 U/ml                                                          0.0                                                   ______________________________________                                         K = angiokinase extract                                                  

As shown in the table, purified angiokinase shows increasingfibrinolytic activity as the plasminogen concentration increases.However, the same buffer with no angiokinase shows almost no activity,independent of the plasminogen concentration.

Angiokinase's plasminogen activation is confirmed by the polyacrylamidegel incubation and electrophoresis experiments described under 4.

FIG. 5 shows the results of the experiments on the plasminogenactivating action of the purified extract, with short incubation periodsand in the presence of 20% glycerine (7.5% polyacrylamide in the gel).In FIG. 5, gel A shows the plasminogen incubated with glycerine only.Under these conditions, there was no spontaneous activation; theplasminogen is present as a protein chain with a molecular weight around90000 dalton. The changes in the molecule after incubation withurokinase are shown by gel B. Three molecular components (arrows) may beobserved with molecular weights of 60000, 20000 and 10000 dalton. Gel Cshows the changes in the plasminogen molecule after incubation with thepurified extract according to the invention. There is a clear differencefrom gel A, with plasminogen only. In any case, a considerable portionof the plasminogen remained unchanged after incubation for 30 minutes.

FIG. 6 reports the results of analogous experiments with long incubationperiods. In this figure, gel 1 shows plasminogen incubated at 4° C. fortwo hours. Only the protein chain of the native plasminogen is visible.

Gel 2 shows plasminogen incubated for two hours at 37° C. Theplasminogen was partially converted to plasmin. The three bandscorresponding to molecular weights of 90000 (plasminogen), 60000 (heavyplasmin chains) and 20000 dalton (light plasmin chains) are visible.

Gel 3 corresponding to urokinase incubated with plasminogen under thesame conditions shows complete activation. There are other bands showingautocatalytic activity on the part of the plasmin formed.

Gel 4 shows plasminogen incubated with the active principle according tothis invention: the result is similar to that for urokinase, andactivation is more complete than for gel 2 (plasminogenauto-activation). The protein quantities of urokinase and purifiedangiokinase extract were so small that they gave no band in theelectrophoretic gel (gel 5).

These results show that the active principle of the angiokinase extractaccording to this invention attacks plasminogen with the formation ofplasmin activity While the molecular modifications induced are similarto those caused by urokinase, the reaction is slower. Plasminogenauto-activation occurs more rapidly in the presence of purifiedangiokinase extract.

The preceding description of the preparation procedure mentioned amethod for measuring the fibrinolytic activity of the fractionsextracted, as an index of the presence of angiokinase extract in thefraction.

The titration of the fibrinolytic substance isolated using the procedureabove which is the object of the present invention is carried out asbelow, based on the fibrinolytic activity on fibrin plates.

TITRATION OF PURIFIED ANGIOKINASE

Materials: sterile plastic plates, inner diameter 93 mm; humanfibrinogen (Immuno) prepared with fractionation according to Cohn; TRISbuffer, pH 7.8; thrombin (Topostasin, Roche).

Method: 17 ml of a 0.1% human fibrinogen solution in pH 7.8 TRIS bufferis placed on the plate and coagulated with 0.04 ml of thrombin (15NIHU/ml of physiological saline). Under these conditions, a 2.5 mm thickfilm of fibrin is formed (2.503).

The activity of the isolated and purified angiokinase is measured bydrawing 30 μliters with a precision pipet and placing it on the surfaceof the fibrin film to be incubated for 16 hours at 37° C. in athermostat, while held perfectly horizontal.

A fibrinolytic unit (angiokinase unit, AU) is defined as the quantity ofenzyme which causes a circular area of lysis 3.908 mm in diameter onplates of human fibrin, after 16 hours of incubation at 37° C.

This degree of lysis expressed as volume corresponds to the dissolutionof a fibrin clot 0.1% in volume compared to the 30 μliters volumecorresponding to the volume of purified angiokinase extract placed incontact with the fibrin.

Another widely known test is the euglobulin lysis time, which can beused to determine the concentration of modified angiokinase extract.Said euglobulin lysis time may be related to the AU value as defined forthe preceding titration method.

ENGLOBULIN LYSIS TIME Materials and methods

Male Wistar rats are used, weighing 200±5 g.

Blood samples were drawn from the open heart, under mild etheranesthesia, into silicon glass or plastic tubes.

3.8% sodium citrate was used as an anticoagulant in a 1:9 ratio to theblood. As soon as it was drawn, the blood was centrifuged at 2000revolutions per minute for 15 minutes in a refrigerated centrifuge, soas to hold the temperature below +5° C.

The plasma so obtained was diluted with distilled water at +5° C. in thefollowing ratio:

0.5 ml of plasma

9.0 ml of distilled water

0.1 ml of 1% acetic acid.

The mixture was held at +4° C. for 30 minutes, then centrifuged at 2000revolutions per minute for 5 minutes in a refrigerated centrifuge. Afterthe supernatant was removed, the residue was taken up in 1 ml of pH 7.8phosphate buffer and placed in a thromboelastographic cuvette.

Triple Hartert-Hellige thromboelastograph cuvettes were used, in each ofwhich 0.3 ml of englobulin solution was introduced using a highprecision pipette. Exactly one minute later, coagulation was initiatedwith 0.06 ml of 1.29% calcium chloride. The piston was immediatelylowered in each cuvette, and the coagulum was covered with a layer ofliquid paraffin to being recording of the elastogram.

At the end of the curve, the lysis time is calculated along thelongitudinal axis, from the point where the branches diverge 1 mm untilthe point where they converge 1 mm. This measurement is then convertedto minutes based on the chart speed.

The animals were treated intraperitoneally 30 minutes before theintracardiac sampling.

The results of the test are reported below in table III, in proportionto the units of angiokinase.

                  TABLE III                                                       ______________________________________                                        Euglobulin lysis times per angiokinase unit                                                        Lysis time %                                                     UA/kg bodyweight                                                                           (minutes)  activity                                      ______________________________________                                        Controls  --             85         --                                        Angiokinase                                                                             1,000          74         12.9                                      "         3,000          59         30.5                                      "         10,000         42         50.0                                      ______________________________________                                    

PHARMACEUTICAL APPLICATIONS

On the basis of the results obtained, the present invention involves theuse of the purified angiokinase extract, that is, of the angiokinaseenzyme, in parenteral (for example, intravenous, intraarterial andintramuscular ampuls) and enteral pharmaceutical formulations (forexample, gelatin capsules or tablets for oral administration). Ofcourse, these examples do not limit the present invention in any way,since it extends to other possible pharmaceutical formulations andadministration routes.

CLINICAL TRIALS

Administration of angiokinase in human therapy has been shown to be freeof local or systemic secondary effects. Given its excellent tolerance,angiokinase may also be given in loco, while the oral route should beused preferably for prevention or maintenance of results achieved viaother routes.

Angiokinase is to be used clinically for the prevention and treatment ofall conditions of abnormal fibrin formation and thrombus formation, invarious sites and localizations. The following examples of itstherapeutic indications are exemplifying only and do not limit thesignificance of the invention in any way.

Athero-arteriosclerosis at various levels (cerebral, coronary,peripheral) and related thrombosis phenomena (cerebral, coronary,peripheral).

Myocardial infarction.

Cerebral infraction.

Diabetic vasculopathies.

Arteriosclerotic and diabetic retinopathy; thrombosis of variousretinas.

Venous thrombosis; thrombophlebitis.

Post-operatory thrombosis.

Thrombo-embolic disease; pulmonary embolism.

With regard to the various therapeutic dosages, the posology ofangiokinase varies depending on the use (prophylactic or therapeutic)and the indications mentioned above, according to a general schemeexemplified as follows:

(A) Prophylaxis

From 10000 to 50000 AU daily, depending on the case.

(B) Artherosclerotic disease in its various localizations Diabeticretinopathy

From 50000 to 200000 AU daily, broken up into various doses during theday.

(C) Thrombosis and thromboembolisms in progress

From 200000 to 1-2 milion AU daily, broken up into various doses duringthe day.

In cases listed in A and B above, treatment may be continued by thephysician, without necessarily requiring blood coagulative control.

In cases listed in C, however, the treatment period should be limited,as a function of the progress in the individual case and the physician'sopinion. Blood coagulation controls are recommended.

EXAMPLE V Clinical trials with angiokinase

70 patients with athero-arteriosclerotic disease were treated: 26 casesof coronary, 18 of cerebral, 10 of retinal and 20 of peripheral (lowerlimbs). The treatment period was in general 30-35 consecutive days, andangiokinase (AK) was used in daily doses of 90000 AU.

Coronary vasculopathies

Pronounced and often rapid attenuation of painful symptomatology wasobserved in a significant number of cases, involving stenocardiac painand strain-induced dyspnea. Working capacity and physical exercisepossibilities were clearly enhanced.

Of the objective symptomatology, a sharp reduction of arrythmia, up toits disappearance, was noted. Electrocardiograph modifications wereobserved leading to improved coronary irrigation (for example,attenuation or disappearance of S-T tract dyslevel and of extrasystolicphenomena).

In those cases where before treatment, stress tests led to theappearance or worsening of signs of coronary ischemia and arrythmia,treatment with AK led to a marked reduction or even disappearance ofthese phenomena.

Cerebral vasculopathies

On the level of subjective symptomatology, the modifications observedwere particularly significant both in degree and consistency.

A generally rapid improvement was noted in tone and mood, with markedattenuation or disappearance of the sense of disorientation, dizzinessand headache, with improved memory and working capacity.

For the objective symptomatology, cases of recent or relatively recentmotor deficit or sensitive disturbances showed a reduction or evencomplete disappearance of said manifestations, with a cause and effectrelationship.

Ateriosclerotic retinopathy

Examination of the ocular ground at the end of treatment showedpronounced improvement in the retina condition (more evident andcomplete in the most recent cases), with disappearance of themicrothrombosis, absorption of exudates and renewal of visual acuity toa completely satisfactory level.

Periperhal vasculopathies

Sharp regression, even up to complete disappearance, of the claudicationwas observed after the end of treatment. This complete disappearance wasalways observed in second stage cases.

Of the objective symptomatology, improved circulation in the affectedareas was observed in a significant number of cases: more reasonablecolor and temperature returned to said area with noticeable improvementor regression of any trophic disturbances present (ulcerations, turbidsores), and at times with returned pulse in the pedal artery.

EXAMPLE VI Laboratory and instrumental tests in the clinical trials

The following laboratory test results were obtained for the casestreated and observed in example V.

Cholesterol and lipid levels were affected only slightly in anon-significant way.

However, the blood coagulative area constantly showed a sharp andsignificant modification due to:

(a) the reduction or disappearance of the thrombophil condition, withusually unimportant lengthening of coagulation times;

(b) the normalization of the partial thromboplastic time;

(c) the considerable shortening of the lysis time (dilute blood lysistime, englobulin lysis time).

Before treatment, these times were more or less noticeably lengthened.Treatment with AK brought them back to normal values, or at leastshortened them.

In almost all cases where they were measured, the oscillograph resultsshowed sharp improvement, even up to the point of normalization of theoscillation amplitude.

Acute thrombosis

AK was also studied in cases of acute thrombosis attacks, that is, inten cases of acute myocardial infarction, in 5 of cerebral thrombosis,and in 3 of peripheral thrombosis (femoral level).

Progress in all these cases of particular doctrinal and practicalinterest showed clearly the effective lysis activity, in terms of usefulmodifications in the blood coagulative area and in the clinicalcondition.

In these cases of acute thrombosis, the posology of AK was certainlyhigher than that used in example V: from a minimum of 250000 AU to amaximum of 1000000 AU daily, depending on the case and its progress. AKwas administered intravenously or by phleboclysis.

Acute myocardial infarction

Pronounced and rapid improvement was noted in the clinical condition andpain symptomatology, with regression of the condition of collapse andrapid disappearance or marked reduction of arrythmia phenomena.

Progress in the cases treated was completely satisfactory, withsignificant and rapid modifications in the electrocardiograph condition,especially with regard to the S-T portion, the Q wave and the terminal Twave. More precisely, a regression in the dyslevel of the S-T portionwas observed. This is well known to be an expression of lesion potentialin the myocardial fibers in the perinfarcted area. Significantregressions were also observed in the characteristic alterations of theQ-R-S complex and in the negativity of the T wave.

On the level of enzymatic activity (transaminase, creatinphosphokinase),its rapid attenuation or disappearance was observed.

With regard to the clinical, instrumental and laboratory progressobserved in these cases of acute myocardial infarction treated with AK,it should be emphasized that such progress is usually not observed withthe therapies available prior to this invention.

Cerebral thrombosis

In two cases, treatment with AK afforded a rapid return to normalconditions of motility and sensitivity in the affected hemisphere. Saidreturn began 24-48 hours after the beginning of treatment, and developedwithin one week.

The other three cases showed a satisfactory although incompleteregression of the effects on the hemisphere involved.

Peripheral thrombosis, fermoral thrombosis

All three cases showed complete disappearance of the occlusion symptomswithin 24-48 hours, with return to normal circulation in the ischemizedareas, with returned pulse and oscillations.

It should be emphasized that none of the cases treated according to thisinvention showed any signs of hemorrhage, in the injection site oranywhere else. This includes the initially acute cases who received thehighest dosage (1000000 AU daily).

Regular urinalysis revealed no hematuria. Tests of vasal fragilityshowed no pathological variations.

Therefore, based on the clinical and laboratory results for all thecases treated with angiokinase, even those at the highest dose, not onlywas a markedly antithrombophillic condition initiated, but asatisfactory condition of blood coagulative homeostasis was alsomaintained. No hemorrhagic situations were ever encountered.

These results, together with its undisputed antithrombotic activity,good manageability and therapeutic reliability, emphasize the usefulnessof the new substance angiokinase, according to this invention, ascompared with other previously known therapies.

By the very nature of the invention, the comparison can only beindirect, leaving to each practitioner to decide on a case-by-case basiswhether to apply the treatment covered by this invention rather thanpreviously known ones.

We claim:
 1. A pharmaceutical composition for the prophylaxis andtreatment of abnormal fibrin formation and thrombus formation comprisinga pharmaceutical carrier and an effective amount of an active principleknown as angiokinase enzyme isolated by extraction of vascular andrichly vascularized connective tissues from mammals, through enzymaticlysis, with a protein N level of 0.2 μg/ml as measured with Lowry'smethod, as well as a mean molecular weight of approximately 18000dalton; said principle having a fibrinolytic effect on fibrin both inthe presence and absence of plasminogen, where said fibrinolyticactivity is such that 30 μliters of angiokinase extract form a circularlysis area 3.908 mm in diameter on plates of human fibrin 2.503 mmthick, after 16 hours incubation at 37° C.
 2. Pharmaceutical compositionfor the prophylaxsis and treatment of abnormal fibrin formation andthrombus formation in human subjects, athero-arteriosclerosis at variouslevels, myocardial infarction, cerebral infarction, diabeticvasculopathies, arteriosclerotic and diabetic retinopathy, thrombosis ofthe retinal vessels, venal thrombosis, thrombophlebitis, post-operatorythrombosis, thromboembolism disease, pulmonary embolism, comprising atherapeutically effective quantity of the fibrinolytic active principleangiokinase as claimed in claim 2 for oral or parenteral administration,in a dose ranging from 10000 to 2000000 AU, as well as pharmaceuticallycompatible excipients.
 3. A pharmaceutical composition according toclaim 1, for the prophylaxis of abnormal fibrin formation and thrombusformation in a dose ranging from 10000 to 50000 AU daily.
 4. Apharmaceutical composition according to claim 1, for the treatment ofatherosclerotic disease or diabetic retinopathy in a dose ranging from50000 to 200000 AU daily.
 5. A pahrmaceutical composition according toclaim 1, for the treatment of thrombosis and thromboembolisms inprogress, in a dose ranging from 200000 to 2000000 AU daily. 6.Pharmaceutical composition according to claim 5, in the form ofinjectable ampul for parenteral administration or of capsules andtablets for oral administration.
 7. A method of treating abnormal fibrinformation and thrombus formations in human subjects which comprises theadministration of a therapeutically effective amount of a composition asclaimed in claim
 5. 8. A method as claimed in claim 7, wherein saidabnormal fibrin formation and thrombus formation isathero-arteriosclerosis at various levels, myocardial infarction,cerebral infarction, diabetic vasculopaties, atherosclerotic anddiabetic retinopathy, thrombosis of the retinal vessels, venalthrombosis, thrombo-phlebitis, post-operatory thrombosis,thromboembolism disease or pulmonary embolism.